Specific Aims ... Aim 1 ... The focus of this aim will be o

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Project 3SIV models of neurobehavioral,
antiviral, and immunomodulatory SP antagonist(s)
effects

• Andrew A. Lackner, DVM, PhDKate Baker, PhD
• Tulane National Primate Research Center
• Tulane University Health Sciences Center

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Project goals

• To determine the association between SIV
  progression, SP level, and indices of
  psychological disturbance.
• Identify mechanisms whereby SP or SP antagonists
  affect SIV disease progression.

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Hypothesis

• SP production will be increased in SIV-infected
  macaques and SP antagonists will have a
  protective effect on disease progression and
  neurobehavioral parameters by
• Decreasing SIV replication in monocyte/macrophages
  , particularly in the CNS.
• Blocking the known neurobehavioral effects of SP.

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Specific Aims

• Determine the cellular distribution of SP and its
  receptor in tissues of normal and SIV-infected
  macaques at various stages of disease.
• Characterize the natural history of SP in normal
  and SIV-infected rhesus macaques in relation to
  viral, immunologic and behavioral measures.
• Examine the effects of SP antagonists in normal
  and SIV-infected macaques.
• Compare disease progression between continuous SP
  antagonist dosing with intermittent treatment (or
  periodically increased dose), timed to social
  rearrangements.

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Aim 1

• Determine the cellular distribution of SP and its
  receptor in tissues of normal and SIV-infected
  macaques at various stages of disease.
• Existing data on the distribution of SP and its
  receptor in nonhuman primates is primarily
  limited to neurons. The focus of this aim will
  be on cells of the immune system, particularly
  monocyte/macrophages in the CNS.
• Mechanisms of SP and its antagonists in AIDS
  pathogenesis can not be discerned unless the
  relevant cell types are defined in vivo.
• The study will be done using archival tissues
  collected from SIV-infected macaques and matched
  controls.

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Aim 1 Determine the cellular distribution of SP
and its receptor in tissues of normal and
SIV-infected macaques at various stages of
disease (acute infection, clinically
asymptomatic, terminal)
Archival tissues from 24 infected and 24
uninfected macaques 8 acute infection 8
matched controls 8 clinically asymptomatic/viral
set point 8 matched controls 8 terminal AIDS
8 matched controls
Detection of SP cells by IHC and ISH
Detection of NK1R cells by IHC and ISH
Multiparameter confocal microscopy for cell type
and virus (for SIV-infected animals)
SP NK1R Macrophage
SP NK1R NK cell
SP NK1R T cell
SP NK1R SIV
Examination of SP and its receptors will focus on
examining macrophages and T cells which may
support viral replication and/or be involved in
the adaptive or innate immune response (including
NK cells). Multiple tissues will be examined but
the focus will be on the CNS. Examination of
cell type will be performed in the context of
histopathologic lesions.
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Aim 2

• Characterize the natural history of SP in normal
  and SIV-infected rhesus macaques in relation to
  viral, immunologic and behavioral measures.
• Prospectively examine i) levels of SP in blood
  and CSF over time, ii) viral load, iii) humoral
  and cellular immune responses and iv) natural
  killer cell activity.
• Prospectively examine circadian rhythms and
  behavioral changes focusing on indices of
  psychological disturbance.

This aim will establish the relationship between
SP, viral load, behavior and immunologic
parameters.
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Aim 3

• Examine the effects of SP antagonists in normal
  and SIV-infected macaques
• Evaluation of the pharmacokinetics of SP
  antagonists.
• Evaluate the effects of SP antagonists (for 6
  months) in SIV-infected macaques for effects on
  viral load, viral dynamics and immunologic
  response compared to controls.
• Evaluate the effects of SP antagonists on
  behavior.
• Evaluate the effects of SP antagonists plus
  antiretrovirals (eg tenofovir) in SIV-infected
  macaques.

This aim will be used to determine the
pharmacokinetics, safety and potential efficacy
of SP antagonists in macaques and serve as a
guide for studies in HIV-infected humans
described in project 4.
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Aim 2 Characterize the natural history of SP in
normal and SIV-infected rhesus macaques in
relationship to viral, immunological, circadian,
and behavior measures
All subjects (N32), single housing
All subjects (N32) introduced into pair housing
SP, immunological, and behavioral measures
24 subjects inoculated with SIV
Longitudinal SP, viral, immunological, and
behavioral measures
These data will serve as the baseline data for
subsequent within-animal paired comparisons
following infection and/or treatment with SP
antagonists, as well as contributing data on
normal untreated animals for Aim 2
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Aim 3a Evaluation of the pharmacokinetics of SP
antagonists
8 SIV-infected macaques (released from other
studies)
8 normal uninfected macaques
Treat with SP antagonists
Treat with SP antagonists
Collect blood to measure -Antagonist -Viral
load (for infected animals) -Serum chemistries

• will utilize 8 of the animals shown used in
  aims 2/3 either prior to infection or from the
  uninfected control group

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Aim 2 Characterize the natural history of SP in
normal and SIV-infected rhesus macaques in
relationship to viral, immunological, circadian,
and behavior measures
All subjects (N32), single housing
All subjects (N32) introduced into pair housing
SP, immunological, and behavioral measures
24 subjects inoculated with SIV
Longitudinal SP, viral, immunological, and
behavioral measures for 6 months
These data will serve as the baseline data for
subsequent within-animal paired comparisons
following infection and/or treatment with SP
antagonists, as well as contributing data on
normal untreated animals for Aim 2
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Aim 3d Evaluate the effects of SP antagonists
plus antiretrovirals (eg tenofovir) in
SIV-infected macaques on viral load, immunologic
response and behavior compared to SIV-infected
macaques treated with SP antagonists alone.
16 SIV-pos. subjects Treated with SP antagonist
8 SIV-pos. subjects Treated with SP antagonist
8 SIV-pos. subjects Treated with SP antagonist
plus tenofovir
Longitudinal SP, viral, immunological, and
behavioral measures
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Schedule of animal monitoring
General health check, food consumption and stool
character are recorded daily
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Kinetics of SIV-specific CTL responses in early
SIV infection
Gated through CD3CD8HI lymphocytes
Intestinal lymphocytes
Peripheral blood lymphocytes
mm352 mm353 mm356
mm352 mm353 mm356
0.2
0.1
0.1
0.0
0.0
0.0
7 days p.i.
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3.1
4.1
17.3
10.5
8.7
14.7
14 days p.i.
1.8
5.3
1.3
1.8
3.4
11.4
21 days p.i.
4.6
11.3
8.5
3.0
4.5
22.3
63 days p.i.
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Mamu-A01/p11C, C-M
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SIV-gag tetramer in the CNS parenchyma of an
animal with SIVE
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Elevations in SP in humans infected with HIV and
macaques infected with SIV
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Decreased NK cell activity during SIV infection
LU20/107PBMC
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Specific Aims

• Determine the cellular distribution of SP and its
  receptor in tissues of normal and SIV-infected
  macaques at various stages of disease.
• Characterize the natural history of SP in normal
  and SIV-infected rhesus macaques in relation to
  viral, immunologic and behavioral measures.
• Examine the effects of SP antagonists in normal
  and SIV-infected macaques.
• Compare disease progression between continuous SP
  antagonist dosing with intermittent treatment (or
  periodically increased dose), timed to social
  rearrangements.

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The macaque model is ideally suited to this
project

• The rhesus macaque is the premier animal model
  for the study of AIDS neuropathogenesis.
• The rhesus macaque has a long history of being
  employed to model psychopathology -- a
  well-characterized model of indices of
  psychological disturbance.

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Neurobehavioral measures

• Telemetry data circadian rhythms, sleep,
  activity level
• Behavioral observation
• Species-appropriate social and non-social
  behavior
• Abnormal behaviors
• Psychological indices of disturbance

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Primate models of psychological disturbance
Depressive behaviors

• Operational Definition
• Slumped or collapsed body posture
• Periods of unresponsivity to environmental
  stimuli
• Periods of hypervigilance
• Ethopharmacologic validation
• Amelioration associated with tricyclics and MAO-I
  inhibits
• Exacerbated with reserpine
• Construct validation (causes and physiological
  correlates)
• Associated with social stress as in humans
• Associated with altered HPA and dopaminergic
  function as in humans
• Face validity Observational characteristics in
  humans
• Slumped or collapsed body posture
• Unresponsivity to environmental stimuli

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Primate models of psychological disturbance
Anxiety-related behaviors

• Operational Definition
• Self-directed displacement behaviors
• Scratching
• Self-grooming
• Body shaking
• Ethopharmacologic validation
• Amelioration (dose dependent) with lorazepam,
  midazolam, diazepam, clonidine
• Exacerbated with axiogenics (FG142, b-CCE)
• Construct validation (causes and physiological
  correlates)
• Associated with uncertainty and anticipation of
  aversive conditions as in humans
• Associated with autonomic hyperarousal as in
  humans
• Face validity Observational characteristics in
  humans
• Self-directed displacement behaviors
• Scratching
• Self-grooming

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Aim 2 Characterize the natural history of SP in
normal and SIV-infected rhesus macaques in
relationship to viral, immunological, circadian,
and behavior measures
All subjects (N32), single housing
All subjects (N32) introduced into pair housing
SP, immunological, and behavioral measures
24 subjects inoculated with SIV
SP, viral, immunological, and behavioral measures
To aim 4
8 SP antagonist plus Tenofovir after 6 mo.
8 continued treatment with SP antagonist
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Aim 4 Compare disease progression in stable and
unstable social settings, and compare continuous
SP antagonist dosing with intermittent treatment
(or periodically increased dose), timed to social
rearrangements. Social stress, indices of
psychological disturbance, and disease progression

• Psychosocial stressors accelerates progression of
  HIV disease in humans
• Social disruption (introductions and separations)
  accelerates SIV disease progression in macaques
• Social disruption alters HPA axis and immune
  function in primates.
• Social stress cannot be experimentally
  manipulated in humans.
• The efficacy of targeting SP antagonists to
  periods of heightened stress represent an
  elaboration to the macaque model toward clinical
  trials in humans.

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Aim 4 Compare disease progression in stable and
unstable social settings, and compare continuous
SP antagonist dosing with intermittent treatment
(or periodically increased dose), timed to social
rearrangements.
All subjects (N24), single housing
SP, immunological, and behavioral measures
All subjects (N24) introduced into pair housing
SP, immunological, and behavioral measures
All subject (N24) inoculated with SIV, all
pairs rearranged every 2 months
8 Untreated
8 treated with consistent dose of SP antagonist
8 treated with intermittent or variable dose,
timed to social rearrangements
Longitudinal SP, viral, immunological, and
behavioral measures
These data will serve as the baseline data for
subsequent within-animal paired comparisons
following infection and/or treatment with SP
antagonist, as well as contributing data on
normal untreated animals Eight of these animals
(SIV-neg controls treated with SP antagonist)
will be from aim 3 after a washout
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Interaction/synergy between Project 3 and other
projects and cores
Project 3

• Project 1 and 2
• Selection of SP antagonist
• Relevant cell types to examine
• Antiviral and immunomodulatory effects
• Project 4
• Objectives of projects 3 and 4 are complementary
• Core B
• Evaluate SP antagonists for effects on macaque
  cells and SIV
• In vitro studies will inform dose for macaque
  studies
• Core C
• Pharmacokinetics
• Statistical analysis
• Study design

• Project 1 and 2
• In vivo relevance of in vitro data
• Project 4
• Safety
• Long term virologic and immunologic effects
• Survival
• Guide future clinical studies

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SIV disease course
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Cytokines
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Intracellular cytokine staining to examine immune
function in situ
IFNg CD3 CD68
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Conclusions

• Neuroinvasion by pathogenic SIV occurs within 14
  days of infection
• Peak vireimia
• Upregulation of endothelial adhesion molecules,
  chemokines and cytokines
• Primary cell type productively infected is the
  perivascular macrophage (subsets?)
• Virus specific CTLs appear in the CNS coincident
  with declines of viral load in the CNS
• Neuroinvasion is associated with evidence of
  neuronal dysfunction

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500
P .014
400
300
LU20/107 PBMC
200
100
0
SIV (n 3)
SIV (n 8)
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HIV, Substance P, and Psychological Disturbance

• HIV is neurovirulent
• Impairs fine motor control, memory, emotional
  control.
• Causes motor slowing, sleep disturbance
• Results in dementia in 25 of cases
• Associated with high rates of depressive and
  anxiety symptoms
• SP plays a role in depression and anxiety
• Rodent model demonstrates relationship between
  exogenous SP, stress paradigms, and SP levels
• Humans elevated SP associated with the
  expression of depressive and anxiety-related
  behaviors NK1 antagonists ameliorate these
  symptoms

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The SIV/macaque model of neuroAIDS

• Neuropathology very similar to that seen in
  HIV-infected humans
• 25 to 50 of SIV-infected macaques develop SIVE.
  Greater incidence in rapid progressors
• SIV is present in the lesions, primarily within
  cells of monocyte/macrophage lineage
• Neuroinvasion occurs within 7 to 14 days of
  infection coincident with
• peak viremia
• Increased numbers of perivascular macrophages
• Early changes associated with SIV infection
  include altered circadian rhythms in body
  temperature and motor activity
• All pathogenic strains of SIV are neuroinvasive
• Development of macrophage-tropism is necessary
  but not sufficient for development of SIVE

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Aim 2 Characterize the natural history of SP in
normal and SIV-infected rhesus macaques in
relationship to viral, immunological, circadian,
and behavior measures Aim 3 Examine the effect
of SP antagonists in normal and SIV-infected
macaques
All subjects (N32), single housing
SP, immunological, and behavioral measures
Aim 3b and 3c
All subjects (N32) introduced into pair housing
Aim 2
SP, immunological, and behavioral measures
24 subjects inoculated with SIV
8 SIV-neg. subjects Treated with SP antagonist
Longitudinal SP, viral, immunological, and
behavioral measures for 6 months
To aim 4
These data will serve as the baseline data for
subsequent within-animal paired comparisons
following infection and/or treatment with SP
antagonists, as well as contributing data on
normal untreated animals for Aim 2